MECHANISM OF ACTION OF CYSTATHIONINE SYNTHASE

Cystathionine synthase [rat liver] catalyzes the formation of cystathionine from serine, as well as .beta.-chloroalanine, and homocysteine. The stereochemistry of the replacement of the .beta.-OH group of serine by homocysteine was established by converting cystathionine, derived from 2S, 3R-[3-3H]serine, to cysteine with .gamma.-cystathionase. This conversion does not alter the configuration at the .beta. carbon of cystathionine. With previously used procedures, it was determined that the cysteine so obtained was 2R, 3R-[3-3H]cysteine, hence the displacement of the OH group of serine by homocysteine proceeds with retention of configuration. The kinetics of the replacement reaction show an intersecting line pattern. This result, together with the observation that in the absence of homocysteine, little, if any, catalytic conversion of .beta.-chloroalanine to serine occurs, indicates that the .beta. substituent is not released from the enzyme prior to binding of homocysteine. In the absence of homocysteine, the enzyme catalyzes the exchange of tritium from [.alpha.-3H]serine and .beta.-[.alpha.-3H]chloroalanine with solvent protons. At pH 7.8, the rate of this exchange reaction is nearly equal to Vmax of cystathionine formation. The rate of pyruvate formation from serine or .beta.-chloroalanine in the absence of homocysteine is maximally 2% that of the tritium exchange rates. In the absence of homocysteine, cystathionine synthase catalyzes .alpha.-proton abstraction more rapidly than elimination of the .beta.-substituent. Several mechanisms are proposed by which elimination of the .beta. substituent from the substrate-derived carbanion is prevented in the absence of homocysteine. Cystathionine synthase is irreversibly inactivated by 30 mM 2-amino-4-chlorobutyric acid with t1/2 = 31 min.